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Thromboxane A2-induced bi-directional regulation of cerebral arterial tone.

ABSTRACT: Myosin light chain phosphatase plays a critical role in modulating smooth muscle contraction in response to a variety of physiologic stimuli. A downstream target of the RhoA/Rho-kinase and nitric oxide (NO)/cGMP/cyclic GMP-dependent kinase (cGKI) pathways, myosin light chain phosphatase activity reflects the sum of both calcium sensitization and desensitization pathways through phosphorylation and dephosphorylation of the myosin phosphatase targeting subunit (MYPT1). As cerebral blood flow is highly spatio-temporally modulated under normal physiologic conditions, severe perturbations in normal cerebral blood flow, such as in cerebral vasospasm, can induce neurological deficits. In nonpermeabilized cerebral vessels stimulated with U-46619, a stable mimetic of endogenous thromboxane A2 implicated in the etiology of cerebral vasospasm, we observed significant increases in contractile force, RhoA activation, regulatory light chain phosphorylation, as well as phosphorylation of MYPT1 at Thr-696, Thr-853, and surprisingly Ser-695. Inhibition of nitric oxide signaling completely abrogated basal MYPT1 Ser-695 phosphorylation and significantly increased and potentiated U-46619-induced MYPT1 Thr-853 phosphorylation and contractile force, indicating that NO/cGMP/cGKI signaling maintains basal vascular tone through active inhibition of calcium sensitization. Surprisingly, a fall in Ser-695 phosphorylation did not result in an increase in phosphorylation of the Thr-696 site. Although activation of cGKI with exogenous cyclic nucleotides inhibited thromboxane A2-induced MYPT1 membrane association, RhoA activation, contractile force, and regulatory light chain phosphorylation, the anticipated decreases in MYPT1 phosphorylation at Thr-696/Thr-853 were not observed, indicating that the vasorelaxant effects of cGKI are not through dephosphorylation of MYPT1. Thus, thromboxane A2 signaling within the intact cerebral vasculature induces "buffered" vasoconstrictions, in which both the RhoA/Rho-kinase calcium-sensitizing and the NO/cGMP/cGKI calcium-desensitizing pathways are activated.

SUBMITTER: Neppl RL

PROVIDER: S-EPMC2649081 | biostudies-other | 2009 Mar

REPOSITORIES: biostudies-other

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Thromboxane A2-induced bi-directional regulation of cerebral arterial tone.

Neppl Ronald L RL Lubomirov Lubomir T LT Momotani Ko K Pfitzer Gabriele G Eto Masumi M Somlyo Avril V AV

The Journal of biological chemistry 20081217 10

Myosin light chain phosphatase plays a critical role in modulating smooth muscle contraction in response to a variety of physiologic stimuli. A downstream target of the RhoA/Rho-kinase and nitric oxide (NO)/cGMP/cyclic GMP-dependent kinase (cGKI) pathways, myosin light chain phosphatase activity reflects the sum of both calcium sensitization and desensitization pathways through phosphorylation and dephosphorylation of the myosin phosphatase targeting subunit (MYPT1). As cerebral blood flow is hi ...[more]

PMID: 19095646

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Project description:BackgroundThromboxane synthase (TxS) metabolizes the cyclooxygenase product, prostaglandin H(2), into thromboxanes. Some of the thromboxanes are known to be biologically active on cancer cells. The aim of the study was to investigate the expression of thromboxane synthases, TBXAS1 and the thromboxane A2 receptor, TBXA2R in a cohort of human breast cancer patients and also to assess their potential clinical relevance.MethodsHuman breast tumour tissues (n = 120) and non-neoplastic mammary tissues (n = 32) were studied. Levels of TBXA2R and TBXAS1 transcripts were quantified using quantitative real-time RT-PCR analysis and correlated with clinical/pathological information including nodal status, grade, prognosis and long term survival (median follow-up period 120 months).ResultsBreast tumour tissue expressed higher levels of TBXA2R compared with normal mammary tissues, although the difference was not statistically significant (p = 0.09). There was no difference between tumour and normal tissues for TBXAS1. However, TBXA2R expression was significantly increased in grade 3 tumours(p = 0.006 vs grade 1), while TBXAS1 was significantly reduced in grade 3 tumours (p = 0.026 vs grade 1 tumours). A similar differential expression pattern was seen in tumours from patients with different prognosis, in that patients with predicted poor prognosis had higher, but not statistically different, levels of TBXA2R, and significantly lower levels of TBXAS1 (p = 0.008). Finally, Kaplan-Meier survival analysis has shown that patients with high levels of TBXA2R had significantly shorter disease free survival (103.8 (79.1-128.5) months) compared with those with low levels (123.7 (112.0-135.3)) months, p = 0.043.ConclusionThromboxane synthases are differentially expressed in human breast cancer. While TBXA2R is highly expressed in aggressive tumours and linked with poor prognosis, TBXAS1 is expressed at significantly low levels in high grade tumours and tumour patients with poor prognosis. TBXA2R thus has a significant prognostic value in clinical breast cancer.

Project description:Activated microglia release pro-inflammatory factors and calpain into the extracellular milieu, damaging surrounding neurons. However, mechanistic links to progressive neurodegeneration in disease such as multiple sclerosis (MS) remain obscure. We hypothesize that persistent damaged/dying neurons may also release cytotoxic factors and calpain into the media, which then activate microglia again. Thus, inflammation, neuronal damage, and microglia activation, i.e., bi-directional interaction between neurons and microglia, may be involved in the progressive neurodegeneration. We tested this hypothesis using two in vitro models: (i) the effects of soluble factors from damaged primary cortical neurons upon primary rat neurons and microglia and (ii) soluble factors released from CD3/CD28 activated peripheral blood mononuclear cells of MS patients on primary human neurons and microglia. The first model indicated that neurons due to injury with pro-inflammatory agents (IFN-?) release soluble neurotoxic factors, including COX-2, reactive oxygen species, and calpain, thus activating microglia, which in turn released neurotoxic factors as well. This repeated microglial activation leads to persistent inflammation and neurodegeneration. The released calpain from neurons and microglia was confirmed by the use of calpain inhibitor calpeptin or SNJ-1945 as well as ?- and m-calpain knock down using the small interfering RNA (siRNA) technology. Our second model using activated peripheral blood mononuclear cells, a source of pro-inflammatory Th1/Th17 cytokines and calpain released from auto-reactive T cells, corroborated similar results in human primary cell cultures and confirmed calpain to be involved in progressive MS. These insights into reciprocal paracrine regulation of cell injury and calpain activation in the progressive phase of MS, Parkinson's disease, and other neurodegenerative diseases suggest potentially beneficial preventive and therapeutic strategies, including calpain inhibition.

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Thromboxane A2-induced bi-directional regulation of cerebral arterial tone.

Publications

Thromboxane A2-induced bi-directional regulation of cerebral arterial tone.

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